Immunology In The Laboratory
The ability to measure accurately
and sensitively different aspects of immunological function is an important part of both experimental and
clinical immunology (see Chapter 44). Some of the most commonly used techniques
in the immunological laboratory (immunological assays) are shown in the figure.
Some techniques, such as the differential blood count, have hardly changed in
over a hundred years. Others, such as flow cytometry and the PCR continue to
evolve at a rapid rate as new technologies are developed. In all cases,
clinical laboratories are making increased use of robotics and sophisticated
computational analysis to automate all aspects of the process, to make it
faster, cheaper and more reliable. The ability to integrate many different
measurements (immunological, haematological, psychological, genetic, etc.)
taken from each patient rapidly and reliably is also driving the development of
‘personalized medicine’, where doctors will be increasingly able to tailor each
treatment precisely to match the needs of individual patients.
Flow cytometry (Fig. 45.1–45.3) is one of the most powerful
techniques in the immunologist’s repertoire. Cells are sucked into a fine jet
of liquid so that they pass rapidly across a beam of one or, in more
sophisticated machines, several lasers. Cells scatter the incoming beam of
light by refraction and reflection. Light scattered through a small angle is
called ‘forward scatter’ and is proportional to the size of the cells. Light
scattered through a 90° angle is called ‘side scatter’ and depends on the
granularity of the cell; e.g. a granulocyte has a much larger side scatter than
a lymphocyte (see Fig. 45.1).
Cells can also be mixed with mixed
with antibodies that bind to specific molecules on the cell’s surface. Each
antibody is linked to a molecule (fluorophore) with the property of absorbing
light of one wavelength and re-emitting it at another. Many such molecules
exist, some originally isolated from marine organisms. Light emitted by each
cell is collected by a series of mirrors and then detected by one of several
photomultipliers and stored on a computer. The precise com- position of the
mixture of cells can then be determined by analysis of their signals. In Fig.
45.2, cells from the thymus are shown as positive for CD4, CD8, both, or
neither. The results can be displayed in the form of a dot plot (Fig. 45.1 and
45.2) in which each cell is represented as a dot, or as a histogram (Fig.
45.3). Results from histogram analyses can be superimposed as in Fig. 45.3,
permitting easy comparisons between healthy and disease samples (e.g. blood
cells from patients with leukaemia as shown in figure).
In a further refinement, cells
binding different antibodies can be collected in separate tubes
(fluorescence-activated cell sorting [FACS]), a powerful tool for isolating
very pure cell populations from a mixture.
Immunofluorescence (Fig. 45.4), in addition to its role in flow
cytometry, can also be applied to histological specimens, commonly to identify
autoantibodies or immune complexes, or metastatic cancer cells invading healthy
tissues. Figure 45.4 shows a kidney from a patient with systemic lupus
erythematosus stained with a fluorescent antibody to IgG, which has bound to
the immune complexes along the basement membrane.
ELISA (Fig. 45.5) The enzyme-linked immunoabsorbent assay is one of the most versatile immunological techniques.
In direct ELISA, a target antigen, e.g. microbial proteins, or human DNA, is
adsorbed on to a plastic surface – typically 96 or 396 small ‘wells’ – allowing
many samples to be tested simultaneously. Diluted samples of serum to be tested
are added, and any antibodies specific for the target antigen will become bound
and immobilized to the plastic surface. Unbound serum components are then
washed off and a ‘second’ antibody, e.g. to human Ig, which has been linked to
an enzyme is added. An enzyme is chosen that converts a colourless substrate to
a coloured product, which can then be measured in a spectrophotometer. Direct
ELISA is often used to detect antibodies to microbes in infection (Fig. 45.5
shows the results of testing different human sera for the presence of
antibodies to HIV) or to self antigens in autoimmune disease.
Sandwich ELISA In another variant, a specific antibody is
first adsorbed to the plastic wells, then the serum or other sample to be
tested, and finally the enzyme-linked second antibody, so as to form an
antibody–antigen–antibody ‘sandwich’. Complement components, cytokines, etc.
can be conveniently assayed in this way. Much effort is being put into improved
ELISA-like protocols that have increased sensitivity and can detect many
components in a single sample (known as ‘multiplexing’), reducing sample size,
speed and cost.
